Synthesis and Evaluation of Hydrolyzable Hyaluronan-Tethered

Nov 1, 2005 - Richard Bernasconi,‡ Marina Slavsky,‡ Sandra Dethlefsen,§ Peter K. Jarrett,§ and Robert J. Miller§,*. Biomaterials Science & Engi...
0 downloads 0 Views 321KB Size
Bioconjugate Chem. 2005, 16, 1512−1518

1512

Synthesis and Evaluation of Hydrolyzable Hyaluronan-Tethered Bupivacaine Delivery Systems Diego A. Gianolio,§ Michael Philbrook,§ Luis Z. Avila,§ Hollace MacGregor,§ Su X. Duan,§ Richard Bernasconi,‡ Marina Slavsky,‡ Sandra Dethlefsen,§ Peter K. Jarrett,§ and Robert J. Miller§,* Biomaterials Science & Engineering, and Drug Discovery, Genzyme Corporation, 500 Kendall Street, Cambridge, Massachusetts 02142. Received August 5, 2005; Revised Manuscript Received October 6, 2005

Local anesthetics are useful for reducing acute pain, but their short duration precludes them from use in solely managing postoperative pain. To prolong the duration of local anesthesia, we conjugated bupivacaine to native hyaluronan (HA) and divinyl sulfone cross-linked Hylan A (Hylan B particles) using a hydrolyzable linker incorporating an imide. Bupivacaine was prepared for conjugation to HA by forming the acryl imide derivative. Separately, the carboxyl group of HA was reacted with nipsylethylamine (NEA) using carbodiimide-mediated coupling to provide HA-NEA that was subsequently reduced with tris(2-carboxyethylphosphine) hydrochloride to yield HA carrying a free sulfhydryl (HA-SH). The HA-bupivacaine conjugate was assembled by reacting HA-SH with acrylbupivacaine. Characterization of the conjugates showed 22% degree of modification by 1 mol of carboxyl. In vitro release studies comparing bupivacaine admixed in HA with bupivacaine conjugated to HA showed half-lives of 0.4 ( 0.1 h, and 16.9 ( 0.2 h, respectively, and the bupivacaine was released chemically unaltered as confirmed by LC-MS. In vivo studies to assess the duration of anesthetic activity were performed in a rat sciatic nerve blockade model. For these studies, bupivacaine was conjugated to Hylan B following a similar procedure, and the degree of modification obtained was 14%. Free bupivacaine (3 and 16 mg/kg) and free bupivacaine (3 mg/kg) admixed with Hylan B particles showed nerve block over 4, 9, and 6 h, respectively. Free bupivacaine (3 mg/kg) admixed with bupivacaine (13 mg/kg) conjugated to Hylan B particles showed a four to 5-fold longer impairment of motor function over the free bupivacaine formulations with a total block time of 19 h. Bupivacaine conjugated to Hylan B particles has the potential to prolong the duration of local anesthesia.

INTRODUCTION

Minimizing postoperative surgical recovery periods has become one of the major goals of hospitals to contain and reduce health care costs (1-3). Optimized local and regional anesthesia regimens improve postoperative rehabilitation by modulating the postoperative stress response thereby improving pulmonary and gastrointestinal function and facilitating postoperative rehabilitation. In treatments such as total knee arthroplasty, accelerating the postoperative convalescence time has been shown to benefit the functional operation outcome while minimizing postoperative complications (4). Given the increasing number of older patients with higher comorbidity, this is of particular therapeutic importance as well as considerable economic implications (5). Continuous techniques of regional anesthesia such as indwelling catheters, continuous infusions and repeated, fractional dosing are used to provide several days of effective postoperative analgesia. While these drug delivery approaches have proven their utility at accelerating postoperative recovery and preventing postoperative adverse outcomes, they are often cumbersome, prone to complications, time-consuming, and costly. For example, epidural catheters require the need for subcutaneous tunneling and fixation of the catheter, bacterial filters, frequent changes of tubing, catheter site protection and * Corresponding author. Tel: + 617-252-7524. Fax: + 617591-5509. E-mail: [email protected]. § Biomaterials Science & Engineering. ‡ Drug Discovery.

monitoring to prevent infection, and training for caretakers (6). External drug delivery devices and fractional dosing regimens are further challenged by the pharmacokinetic profiles of analgesics, as most widely used anesthetic agents are hydrophilic, small molecules that are rapidly distributed and eliminated from the administration site. For example, local infiltration of bupivacaine shows rapid distribution and elimination with peak systemic levels occurring in 30 to 40 min, followed by a decline to insignificant levels during the next 3 to 6 h (7). Therefore, there exists a clinical need for a longacting local anesthetic that can be simply administered to the patient prior to discharge and can provide 1 to 3 days of local pain management (8). In recent years, several studies investigated approaches to prolong the duration of local anesthetics. Lipids and liposomes (9-11), microspheres (12-14), polymeric implants (15, 16), sodium channel toxins (17, 18), butamben formulations (19), and new molecules that target sodium channel subtypes (20) are all examples of formulations that have shown modest extension of anesthetic duration. Of these, the most promising demonstration of long-term motor blockade was achieved using microspheres combining bupivacaine and an antiinflammatory steroid that showed nerve blockade lasting 2 to 7 days, depending on the species, dosing, and site of administration (21). Hyaluronan (HA) is a naturally occurring linear polysaccharide of alternating β-1,3-D-glucuronic acid and β-1,4N-acetyl-D-glucosamine units found in the extracellular matrix and synovial fluid (22). HA is hydrophilic and

10.1021/bc050239a CCC: $30.25 © 2005 American Chemical Society Published on Web 11/01/2005

Hyaluronan-Tethered Bupivacaine Delivery Systems

forms gels of varying viscosity depending on its molecular weight and concentration. HA that is free of contaminating proteins and nucleotide is nonimmunogenic and therefore an ideal candidate for surgical applications that involve sensitive areas, including nerves and muscles. In addition, HA can be modified using a variety of crosslinkers generating novel copolymeric materials and can be used also for tissue engineering (23). As an example, cross-linkers may contain disulfide bonds that can be reduced by dithiothreitol to yield thiol-modified HA that in turn can be reacted with poly(ethylene glycol) diacrylate and other thiol-modified polymers to generate copolymers capable of controlled release of basic fibroblast growth factor (24, 25). Particular interest has been devoted to HA as a potential delivery vehicle for bupivacaine. Prolongation of bupivacaine’s effect by viscous HA formulations was observed in rabbits with the duration of action and pharmacological effects being dependent directly on viscosity (26). During these studies it was stated that the influences of ionic interactions between bupivacaine free base and HA were difficult to assess since viscosity masked the electrostatic feature. HA derivatives (i.e., cross-linked) were also used to prolong the duration of action of bupivacaine. In a recent study an in situ crosslinked HA derivative was tested in rats as a vehicle for bupivacaine. Although cross-linked HA appeared to be a safe and effective means for delivery, the curing process used in this method imposed a need for a rapid injection technique to avoid needle blockage during administration (27). The aim of this study is to investigate the release rate and efficacy of bupivacaine tethered via a reversible covalent bond to HA. We hypothesized that a reversible covalent bond may be more effective in maintaining the drug in the carrier for a longer period of time. We used a cross-linked form of HA that did not require an in situ curing process. It is known that HA cross-linked with divinyl sulfone (DVS) to form the derivative, Hylan B, shows longer residence time at the injection site compared to un-cross-linked HA (28). Thus, bupivacaine was conjugated via a linker containing a hydrolytically labile bond (i.e. imide) to HA. Moreover, a sulfide bond was included in the proximity of the imide to favor the hydrolysis rate in a fashion similar to the one observed when conjugating the drug paclitaxel to poly(ethylene glycol) (29). This report describes a method to prepare the tethered bupivacaine to HA and Hylan B particles. This HAbupivacaine conjugate had a significantly slower release rate compared to free bupivacaine in an infinite sink in vitro release system. The HA-bupivacaine conjugate was compared against free bupivacaine, and bupivacainehylan B particles admixture formulations in a rat model using the sciatic nerve blockade of motor function. EXPERIMENTAL PROCEDURES

Materials and Methods. Low molecular weight HA (130 kDa) was prepared by gamma-irradiation of bacterially fermented HA (30). HA cross-linked with DVS (Hylan B particles) was prepared following a method in the literature (31). Anhydrous solvents and starting materials were purchased from Sigma-Aldrich Co. and used without further purification. 1H and 13C NMR spectra were obtained on a Varian spectrometer (400 MHz) against tetramethylsilane (δ ) 0.0 ppm). Mass spectra and high-resolution mass spectra (HRMS) were obtained on a Applied Biosystems Qstar XL spectrometer

Bioconjugate Chem., Vol. 16, No. 6, 2005 1513

Figure 1. Synthesis of acrylbupivacaine.

with Turbolonspray source. Thin-layer chromatography (TLC) was performed on Silica Gel 60 F254 precoated on aluminum sheets (EM Separation Technology) using CH3OH:CH2Cl2 (1:19). Dialyses were performed using either membrane tubing (7 kDa MWCO, Spectrum) or tangential flow cassette (10 kDa MWCO, Pierce). Phosphatebuffered saline (PBS) contained 137 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer, pH ) 7.3-7.5, and was purchased as a 10-fold concentrate (EMD Chemicals, Inc.) and diluted to a working concentration with deionized water. UV/visible measurements were obtained using a Varian Cary 50 Probe spectrometer. The molecular weight and degree of modification of HA conjugates were determined using size exclusion chromatography and multiangle laser light scattering (SEC-MALLS) analyzer (model DAWN-EOS, Wyatt Technology). A SEC-MALLS analyzer was connected to refractive index and UV/visible (model UVIS-205, Perkin-Elmer) detectors. Separation was performed using a size exclusion column (300 Å, 7.5 × 300 mm, Macrosphere, Alltech), and the mobile phase was a 0.05 M Na2SO4 solution. HA degree of modification was defined as the molar percentage of conjugated repeating dimeric units in HA. The quantitation of bupivacaine rate of release in vitro was determined by high performance liquid chromatography using a C18 reverse-phase column (100 Å, 4.6 × 150 mm, Waters Symmetry) and mobile phase consisting of 55% 10 mM Na2HPO4 in 0.1% H2PO4 and 45% acetonitrile (pH 2.93.0). The purity of bupivacaine released in vitro was determined by high performance liquid chromatography (HPLC model Waters 2690) connected to a UV/visible detector (model Waters 2487) reading at 210 nm and a mass spectrometer (model Waters ZQ-2000) with positive ion electrospray interface. The column used for the separation was a C8 reverse-phase (100 Å, 4.6 × 250 mm, Ace 5, Mac-Mod Analytical). The separation was performed at a column temperature of 25 °C ( 2 °C. Mobile Phase A ) 0.1% HCOOH + 0.01% trifluoroacetic acid (TFA) in water. B ) 0.1% HCOOH + 0.01% TFA 60% CH3CN 20% CH3OH 20% H2O. Flow rate: 1 mL/min split to 0.2 mL/min after the UV/visible detector into the mass spectrometer. A linear gradient was used: 5-100% B over 10 min, followed by 100% B for 5 min. Synthesis of Acryl Bupivacaine (Figure 1). Bupivacaine (1, 500 mg, 1.74 mmol) was placed in a 100-mL round-bottom flask and dried by azeotropic removal of water with toluene under reduced pressure. Anhydrous THF (5 mL) was added under a nitrogen atmosphere. A suspension of 60% NaH in mineral oil (140 mg, 3.48 mmol) was added, gradually allowing for venting. The resulting mixture was allowed to stir for 30 min at room temperature. Acryloyl chloride (157 µL, 1.92 mmol) was subsequently added. The reaction flask was equipped with a condenser and placed in an oil bath, and the reaction mixture was allowed to stir under reflux for 1 h under nitrogen. After completion of the reaction, as monitored by TLC, the mixture was allowed to cool to

1514 Bioconjugate Chem., Vol. 16, No. 6, 2005

Gianolio et al.

Figure 2. Synthesis of HA conjugates. 5a HA-bupivacaine 130 kDa MW, 22% degree of modification. 5b Hylan B-bupivacaine particles, 14% degree of modification.

room temperature and the reaction quenched by addition of MeOH (1 mL). The solvents were removed under reduced pressure, and the crude product was dissolved in dichloromethane (50 mL), transferred into a separatory funnel and washed with water (100 mL). The organic phase was collected, dried with Na2SO4, and concentrated under reduced pressure. An aliquot was further purified by chromatography over silica using 5% MeOH in dichloromethane. The product acryl bupivacaine (2, 162 mg) was obtained as a white solid after removal of solvent under reduced pressure. Rf (methanol/dichloromethane, 1/19): 0.43. 1H NMR (DMSO-d6): δ ) 0.83 (t, 3Hs, J ) 7.6 Hz, CH3 alkyl), 1.2-1.3 (m, 3Hs, CH2), 1.4-1.5 (m, 5Hs, CH2), 1.7-1.8 (m, 2Hs, CH2), 1.9 (s, 3Hs, CH3 aromatic), 2.0 (s, 3Hs, CH3 aromatic), 2.3-2.4 (m, 2Hs, CH2), 5.7 (d, 1H, J ) 10.0 Hz, acryl), 5.8 (dd, 1H, J ) 10.0, 16.0 Hz, acryl), 6.3 (d, 1H, J ) 16.0 Hz, acryl), 7.17.3 (m, 3Hs, aromatic) ppm; 13C NMR (DMSO-d6): δ ) 14.3, 14.5, 18.0, 18.6, 20.6, 21.6, 25.6, 28.7, 29.5, 49.5, 55.4, 63.2, 129.0, 129.5, 132.2, 136.4, 136.5, 136.9, 137.2, 167.2, 167.3 ppm HRMS calculated for C21H31N2O2 (M + H+): 343.2386; found: 343.2386. Synthesis of HA-NEA. 3-Nitro-2-pyridinesulfenylethylamine (NEA) was prepared according to a published procedure (32, 33). To a solution of HA (1.5 g, 3.74 mmol, 130 kDa MW) in water (375 mL) was added 1-hydroxybenzotriazole (1.0 g, 7.48 mmol) followed by NEA (1.5 g, 5.61 mmol). The pH of the reaction mixture was adjusted to 4.5 using 2 N HCl. Subsequently, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.15 g, 11.22 mmol) was added, and the reaction was allowed to stir at room temperature for 2 h. The resulting HA carrying NEA (3) was purified by exhaustive dialysis using a 7 kDa MWCO membrane against PBS, followed by dialysis against water overnight. HA-NEA was recovered by lyophilization as yellow foam (95% yield). Molecular weight and degree of modification of carboxymodified HA-NEA were determined by SEC-MALLS analysis with detection at 350 nm.

Synthesis of HA-SH (Figure 2). Compound 3 (1.0 g, 2.5 mmol, 130 kDa MW, and 22% degree of modification) was treated with a 50 mM tris(2-carboxyethylphosphine) hydrochloride (TCEP) solution (250 mL) at 4 °C for 4 h. The resulting HA carrying free sulfhydryl (4) was purified by exhaustive dialysis using a 7 kDa MWCO membrane against 30 mM succinate buffer containing 120 mM NaCl (pH ) 4.5) followed by dialysis against water overnight. HA-SH was recovered by lyophilization as off-white foam (90% yield). Free sulfhydryl group was quantitated colorimetrically using Ellman’s reagent (34, 35). Synthesis of HA-Bupivacaine (Figure 2). A solution of compound 4 (50 mg, 0.130 mmol of HA, 0.032 mmol of free SH) in water (10 mL) was pH adjusted to 8.0 using triethanolamine and the solution cooled using an ice-water bath. A solution of compound 2 (14 mg, 0.038 mmol) in dimethylformamide (DMF, 130 µL) was added to the solution of 4. Additional DMF (5 mL) was added to maximize the solubility of both compounds 2 and 4. The solution was stirred at 4 °C overnight. The consumption of the free sulfhydryl groups was monitored colorimetrically (Ellman’s reagent). At the end of the reaction, the mixture was transferred into a 7 kDa MWCO membrane and dialyzed at 4 °C against PBS overnight, followed by dialysis against water for 6 h. The retentate was lyophilized to give a white solid product (40 mg, 80% yield). The results of the SEC-MALLS and UV/visible spectroscopy were consistent with the HAbupivacaine conjugate, 5a. Synthesis of Hylan B-Bupivacaine (Figure 2). Bupivacaine was conjugated to Hylan B following a similar procedure to the one described above for compound 5a. The synthesis was performed starting from sterile Hylan B particles and operating under aseptic conditions. Hylan B particles (378 mL, 5.3 mg/mL in 0.9% NaCl solution) were functionalized with NEA and reduced to the free sulfhydryl form. Degree of modification was 14% as determined colorimetrically using Ellman’s reagent (34, 35). Hylan B particles carrying free sulfhy-

Hyaluronan-Tethered Bupivacaine Delivery Systems

Bioconjugate Chem., Vol. 16, No. 6, 2005 1515

Table 1. Scoring Method for the Rat Sciatic Nerve Blockade Study score

observations

2

completely blocked rats dragged their leg and failed to grasp the bars upon elevation of the hindquarters partially blocked rats walked gathering the forepart of the foot, keeping it sideways, and had limited ability to grasp the bars normal rats were able to walk and grasp normally

1 0

dryl group were reacted with 2. The Hylan B-bupivacaine conjugate particles (5b) were transferred into a 7 kDa MWCO membrane and dialyzed at 4 °C against PBS overnight, followed by dialysis against water for 6 h. The retentate was lyophilized to give a white solid product (1.9 g, 94% yield). Degree of modification was 14% as determined by UV/visible spectroscopy with detection at 260 nm, using bupivacaine as a standard. Release Rate of Bupivacaine In Vitro. The release rate of bupivacaine in vitro (infinite sink) from the compound 5a was compared to an admixture of free bupivacaine in a HA solution. To prepare the test sample, compound 5a was dissolved at 5% w/w in distilled water. The admixed control sample was prepared by dissolving one part bupivacaine hydrochloride in 99 parts of a 5% aqueous solution of HA (MW 130 kDa). Aliquots (approximately 1 to 1.5 g) of each solution were weighed in triplicate into tangential flow cassettes (10 kDa MWCO, Pierce). The cassettes were then placed individually in 0.1 M PBS (250 mL) at 37 °C with stirring. The composition of the receptor phase over time was analyzed by HPLC. In Vivo Studies, Sciatic Blockade Technique, and Evaluation. Female Sprague Dawley rats weighing 250 to 300 g were acclimated with free access to food and water for a minimum of 5 days prior to the procedure. Three days prior to each experiment, rats were acclimated to stainless steel cages with mesh flooring. Anesthesia was induced with 2-3% isoflurane in 100% oxygen in a sealed chamber. The skin of the dorsal thigh was shaved, and 200 µL of either a control solution or test formulation was injected into the perineural space of the sciatic nerve. Animals were allowed to recover and were returned to their cages. Immediately following recovery, all animals were observed for the onset of motor block. Motor block was defined as loss of local motor function, exhibited by foot dragging, curling of toes, weight bearing on the lateral margin of the foot, and inability to grab the bars of the cage. The extent of nerve blockade was assessed by monitoring the rats over time using a three-point scoring system (Table 1). The approximate time to onset was recorded. Animals were evaluated every hour until motor block was observed. If no evidence of motor block was observed for more than three consecutive hours, animals were considered unaffected. The experimenter was blinded as to which formulation was injected. Histology. One week after injection, the animals were sacrificed and the sciatic nerves and surrounding tissues were removed, fixed in formalin, and processed for histology by hematoxylin and eosin staining. Slides containing the tissue sections were examined by a board certified pathologist and blinded to the treatment, and microscopic findings were graded according to the intensity and extent of change.

Figure 3. Release profiles of free and conjugated bupivacaine (5a) into receptor phase. RESULTS AND DISCUSSION

Bupivacaine lacks reactive sites (e.g. primary amine, hydroxyl group) for mild chemical modification; however, it possesses an amide bond that could potentially be a site for acylation after deprotonation to give an imide conjugate. The hydrolysis of the imide bond in the resulting HA-bupivacaine conjugate (5a) may either regenerate authentic bupivacaine or produce an undesirable sideproduct depending on which of the two imide bonds undergoes hydrolytic cleavage. With this possibility in mind, we designed the synthesis of the linker to include a sulfide group in proximity to one of the imide functional groups to favor the hydrolysis toward the regeneration of bupivacaine (29). Using this approach described in Figures 1 and 2, bupivacaine was conjugated to HA (5a) with a 22% degree of modification and an overall yield of 80%. To avoid premature hydrolysis and consequent loss of bupivacaine from the conjugate, PBS and water used for dialysis were chilled to 4 °C before use and maintained in the cold room (4 ( 2 °C) during workup. The in vitro release rate of bupivacaine from compound 5a was compared to a physical admixture of bupivacaine in HA (MW 130 kDa). Figure 3 shows the release profiles obtained from this experiment. Bupivacaine hydrochloride admixed to HA quickly eluted into the receptor phase, with half of the drug eluting within 1 h and 100% of the drug eluting within 6 h. In contrast, the HAbupivacaine conjugate (5a) showed more prolonged delivery with half the drug eluting within 17 h and 100% of the drug eluting within 2-3 days. The release profile follows approximately first-order kinetics consistent with the expected pseudo-first-order hydrolysis reaction. LC-MS analysis of the hydrolysis solution from compound 5a showed that 95% of the molecules in the receptor phase were authentic bupivacaine (Figure 4). The remaining peaks present in the chromatogram were attributed to impurities generated during the preparation of the conjugate and were also observed in the control samples without bupivacaine. This result agrees with our initial design hypothesis that the thioether moiety on the methylene β to the acyl group would render this imide bond more susceptible to hydrolysis relative to the other imide bond (Figure 5). The preferential acceleration of the hydrolysis of this bond is due perhaps to a combination of favorable factors. One possible factor is the stabilization of the tetrahedral intermediate by the overlap of low lying empty d orbitals of the sulfur atom with the nonbonded electrons on one of the two oxygen atoms. Another contributing factor that may influence the carbonyl cleavage site is the steric hindrance of the substituents around the two tetrahedral transition states. In particular, inspection of the putative

1516 Bioconjugate Chem., Vol. 16, No. 6, 2005

Gianolio et al.

Figure 4. LC-MS analysis of the hydrolysis solution in the receptor phase. Table 2. Amounts of Free Bupivacaine and Bupivacaine from the Conjugate 5b Injected in Each Rat

saline Hylan B particles admixture Hylan B conjugate particles saline

free bupivacaine (mg/kg)

conjugated bupivacainea (mg/kg)

Nb

3 3 3 16

0 0 13 0

5 10 9 9

a Hylan B-bupivacaine particles (5b) 14% degree of modification. b N ) number of animals for each group.

Figure 6. Nerve blockade scores as a function of time. Scoring methods shown in Table 1. N ) number of animals for each group. Values are means.

Figure 5. 6a, major product of hydrolysis observed. 6b, side product.

tetrahedral intermediate for the hydrolysis of the two carbonyls shows that the tetrahedral intermediate that gives free bupivacaine is less sterically hindered and therefore more stable. This greater stability would lead to a higher equilibrium concentration and in turn faster subsequent hydrolysis rate. These explanations work synergistically to favor the desired hydrolysis. Unmodified HA has a short residence time at most placement sites in the body due to its rapid uptake and degradation by cells (36). To create an HA-based drug carrier that can persist for several days at the placement site we used the insoluble divinyl sulfone (DVS) crosslinked HA. This HA derivative is used in several approved HA products and has a well-established safety profile (37, 38). Thus we conjugated bupivacaine to DVS cross-linked HA, Hylan B particles (31), using the same synthetic approach described for the soluble HA conjugate described above to give the bupivacaine conjugate (5b) with a 14% degree of modification and an overall yield of 94%.

In vivo studies were conducted using an adaptation of the rat sciatic nerve model (39). The duration of efficacy of bupivacaine conjugate (5b) relative to bupivacaine free in solution was assessed in vivo by injecting the conjugate in the proximity of the rat sciatic nerve and by observing the loss and recovery of motor function over time. Control rats were injected with either a 0.5% solution of bupivacaine in saline or an admixture of bupivacaine and Hylan B particles. Table 2 summarizes the amounts of free and conjugated bupivacaine injected per kilogram of animal for each of the controls and test samples. Rats injected with bupivacaine next to the sciatic nerve generally lost their ability to ambulate as a consequence of the nerve blockage. They dragged the injected leg, gathered the forepart of the foot, and lost their grasping ability. The onset time for each rat generally showed nerve blockage within the first 15 min. Figure 6 shows that the duration of motor loss for the bupivacaine control or bupivacaine admixed with Hylan B particles was about 5 h for either sample. This observation is consistent with published findings with similar formulations in the same rat model (40). This result shows that the presence of an insoluble HA gel did not significantly affect the loss of motor function in this model.

Hyaluronan-Tethered Bupivacaine Delivery Systems

Bioconjugate Chem., Vol. 16, No. 6, 2005 1517 LITERATURE CITED

Figure 7. (A) Representative histology of a 5b treated animal and (B) tissue taken from one of the nine treated animals showing extensive regions of vacuolation indicative of dilated axon sheaths. Axon/myelin debris from degenerating axons within the dilated spaces are indicated by the arrow.

Rats injected with a combination of 13 mg/kg of conjugated bupivacaine and 3 mg/kg of free bupivacaine showed a 4- to 5-fold longer impairment of motor function compared to those animals injected with free bupivacaine and free bupivacaine in Hylan B particles. Histological evaluation of each of the injection sites revealed minimal to mild epineurial inflammation with fibroplasia in each of the treatment groups (Figure 7A). The incidence and severity appeared to be somewhat increased in the Hylan B-bupivacaine conjugate particletreated nerves relative to their respective untreated controls. Axonal degeneration characterized by locally extensive regions of dilated axon sheets filled with axon/ myelin debris and associated with inflammatory cell infiltrates and Schwann cell hypertrophy was observed in one of the nine animals (Figure 7B). CONCLUSION

Tethering bupivacaine to HA sustained release in vitro and extended the duration of effect in a sciatic nerve blockage model in vivo. Although nerve blockage was significantly extended by conjugation, there was asymptomatic moderate nerve degeneration in one of the nine animals injected with compound 5b. The extension of motor block with compound 5b is very encouraging, although the axonal degeneration observed in one of the nine animals suggests further evaluation before it is used perineurally. This may not be a concern for local anesthesia at the incision line following surgery. Some degree of inflammation and myotoxicity was also reported for other controlled-release formulations for similar applications (12, 27). This study identifies an additional utility of HA as a drug carrier and deserves further investigation.

(1) Michaloliakou, C., Chung, F., and Sharma, S. (1996) Preoperative multimodal analgesia facilitates recovery after ambulatory laparoscopic cholecystectomy. Anesth. Analg. 82, 44-51. (2) Kehlet, H., and Mogensen, T. (1999) Hospital stay of 2 days after open sigmoidectomy with a multimodal rehabilitation programme. Br. J. Surg. 86, 227-30. (3) Collier, P. E. (1995) Are one-day admissions for carotid endarterectomy feasible? Am. J. Surg. 170, 140-3. (4) Capdevila, X., Barthelet, Y., Biboulet, P., Ryckwaert, Y., Rubenovitch, J., and d’Athis, F. (1999) Effects of perioperative analgesic technique on the surgical outcome and duration of rehabilitation after major knee surgery. Anesthesiology 91, 8-15. (5) Rasche, S., and Koch, T. (2004) Regional anaesthesia versus general anaesthesia-pathophysiology and clinical implications. Anaesthesiol. Reanim. 29, 30-8. (6) Mercadante, S. (1999) Neuraxial techniques for cancer pain: an opinion about unresolved therapeutic dilemmas. Reg. Anesth. Pain. Med. 24, 74-83. (7) Physicians’ Desk Reference (2004) Thomson: Montvale, NJ. (8) Grant, S. A. (2002) The Holy Grail: Long-acting local anaesthetics and liposomes. Clin. Anaesthesiol. 16, 345-52. (9) Dollo, G., Le Corre, P., Chevanne, F., and Le Verge, R. (2004) Bupivacaine containing dry emulsion can prolong epidural anesthetic effects in rabbits. Eur. J. Pharm. Sci. 22, 63-70. (10) Mowat, J. J., Mok, M. J., MacLeod, B. A., and Madden, T. D. (1996) Liposomal bupivacaine. Extended duration nerve blockade using large unilamellar vesicles that exhibit a proton gradient. Anesthesiology 85, 635-43. (11) Malinovsky, J. M., Le Corre, P., Meunier, J. F., Chevanne, F., Pinaud, M., Leverge, R., and Legros, F. (1999) A doseresponse study of epidural liposomal bupivacaine in rabbits. J. Controlled Release 60, 111-9. (12) Kohane, D. S., Smith, S. E., Louis, D. N., Colombo, G., Ghoroghchian, P., Hunfeld, N. G., Berde, C. B., and Langer, R. (2003) Prolonged duration local anesthesia from tetrodotoxin-enhanced local anesthetic microspheres. Pain 104, 41521. (13) Montanari, L., Cilurzo, F., Selmin, F., Conti, B., Genta, I., Poletti, G., Orsini, F., and Valvo, L. (2003) Poly(lactide-coglycolide) microspheres containing bupivacaine: comparison between gamma and beta irradiation effects. J. Controlled Release 90, 281-90. (14) Drager, C., Benziger, D., Gao, F., and Berde, C. B. (1998) Prolonged intercostal nerve blockade in sheep using controlledrelease of bupivacaine and dexamethasone from polymer microspheres. Anesthesiology 89, 969-79. (15) Hasirci, V., Bonney, I., Goudas, L. C., Shuster, L., Carr, D. B., and Wise, D. L. (2003) Antihyperalgesic effect of simultaneously released hydromorphone and bupivacaine from polymer fibers in the rat chronic constriction injury model. Life Sci. 73, 3323-37. (16) Park, E. S., Maniar, M., and Shah, J. C. (1998) Biodegradable polyanhydride devices of cefazolin sodium, bupivacaine, and taxol for local drug delivery: preparation, and kinetics and mechanism of in vitro release. J. Controlled Release 52, 179-89. (17) Kohane, D. S., Yieh, J., Lu, N. T., Langer, R., Strichartz, G. R., and Berde, C. B. (1998) A reexamination of tetrodotoxin for prolonged duration local anesthesia. Anesthesiology 89, 119-31. (18) Kohane, D. S., Lu, N. T., Gokgol-Kline, A. C., Shubina, M., Kuang, Y., Hall, S., Strichartz, G. R., and Berde, C. B. (2000) The local anesthetic properties and toxicity of saxitonin homologues for rat sciatic nerve block in vivo. Reg. Anesth. Pain Med. 25, 52-9. (19) Shulman, M., Harris, J. E., Lubenow, T. R., Nath, H. A., and Ivankovich, A. D. (2000) Comparison of epidural butamben to celiac plexus neurolytic block for the treatment of the pain of pancreatic cancer. Clin. J. Pain 16, 304-9. (20) Porreca, F., Lai, J., Bian, D., Wegert, S., Ossipov, M. H., Eglen, R. M., Kassotakis, L., Novakovic, S., Rabert, D. K., Sangameswaran, L., and Hunter, J. C. (1999) A comparison

1518 Bioconjugate Chem., Vol. 16, No. 6, 2005 of the potential role of the tetrodotoxin-insensitive sodium channels, PN3/SNS and NaN/SNS2, in rat models of chronic pain. Proc. Natl. Acad. Sci. U.S.A. 96, 7640-4. (21) Castillo, J., Curley, J., Hotz, J., Uezono, M., Tigner, J., Chasin, M., Wilder, R., Langer, R., and Berde, C. (1996) Glucocorticoids prolong rat sciatic nerve blockade in vivo from bupivacaine microspheres. Anesthesiology 85, 1157-66. (22) Patti, A. M., Gabriele, A., Vulcano, A., Ramieri, M. T., and Della Rocca, C. (2001) Effect of hyaluronic acid on human chondrocyte cell lines from articular cartilage. Tissue Cell 33, 294-300. (23) Prestwich, G. D., Marecak, D. M., Marecek, J. F., Vercruysse, K. P., and Ziebell, M. R. (1998) Controlled chemical modification of hyaluronic acid: synthesis, applications, and biodegradation of hydrazide derivatives. J. Controlled Release 53, 93-103. (24) Cai, S., Liu, Y., Zheng Shu, X., and Prestwich, G. D. (2005) Injectable glycosaminoglycan hydrogels for controlled release of human basic fibroblast growth factor. Biomaterials 26, 6054-67. (25) Shu, X. Z., Liu, Y., Luo, Y., Roberts, M. C., and Prestwich, G. D. (2002) Disulfide cross-linked hyaluronan hydrogels. Biomacromolecules 3, 1304-11. (26) Dollo, G., Malinovsky, J. M., Peron, A., Chevanne, F., Pinaud, M., Le Verge, R., and Le Corre, P. (2004) Prolongation of epidural bupivacaine effects with hyaluronic acid in rabbits. Int. J. Pharm. 272, 109-19. (27) Jia, X., Colombo, G., Padera, R., Langer, R., and Kohane, D. S. (2004) Prolongation of sciatic nerve blockade by in situ cross-linked hyaluronic acid. Biomaterials 25, 4797-804. (28) Larsen, N. E., Leshchiner, E. A., Pollak, C., Balazs, E. A., and Piacquadio, D. (1995) Evaluation of Hylan B (Hylan gel) as soft tissue dermal implants. Mater. Res. Soc. Symp. Proc. 394, 193-7.

Gianolio et al. (29) Schoenmakers, R. G., van de Wetering, P., Elbert, D. L., and Hubbell, J. A. (2004) The effect of the linker on the hydrolysis rate of drug-linked ester bonds. J. Controlled Release 95, 291-300. (30) Miller, R. J., and Shiedlin, A. U.S. Patent 6,383,344, 2002. (31) Balazs, E., and Leshchiner, A. U.S. Patent 4,605,691, 1986. (32) Calias, P., and Miller, R. U.S. Patent 6,749,865, 2004. (33) Vyas, D. M., Benigni, D., Rose, W. C., Bradner, W. T., and Doyle, T. W. (1989) Synthesis and in vivo antitumor activity of novel mitomycin A disulfide analogs. J Antibiot. (Tokyo) 42, 1199-201. (34) Ellman, G. L. (1959) Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70-7. (35) Riddles, P. W., Blakeley, R. L., and Zerner, B. (1983) Reassessment of Ellman’s reagent. Methods Enzymol. 91, 4960. (36) Vercruysse, K. P., and Prestwich, G. D. (1998) Hyaluronate derivatives in drug delivery. Crit. Rev. Ther. Drug Carrier. Syst. 15, 513-55. (37) Piacquadio, D., Larsen, N. E., Denlinger, J. L., and Balazs, E. (1998) Hylan B gel (Hylaform) as a soft tissue augmentation material. Basic Clin. Dermatol. 15, 269-91. (38) Larsen, N. E., Pollak, C. T., Reiner, K., Leshchiner, E., and Balazs, E. A. (1993) Hylan gel biomaterial: dermal and immunologic compatibility. J. Biomed. Mater. Res. 27, 112934. (39) Hu, D., Hu, R., and Berde, C. B. (1997) Neurologic evaluation of infant and adult rats before and after sciatic nerve blockade. Anesthesiology 86, 957-65. (40) Gerner, P., Mujtaba, M., Sinnott, C. J., and Wang, G. K. (2001) Amitriptyline versus bupivacaine in rat sciatic nerve blockade. Anesthesiology 94, 661-7.

BC050239A